||This work aims to investigate the chemical and biological responses of a-C:H films produced by PECVD technique onto substrate of Ti6Al4V alloy. The films produced were characterized morphologically and structurally by optical microscope (OM), SEM, AFM, EDX, FTIR Raman Spectroscopy (RS) and XPS. Afterwards, biocompatibility and cytotoxicity tests on osteoblastic cells and/or Human Peripheral Blood Mononuclear Cells (PBMCs) were performed on the Ti6Al4V alloy with coated and uncoated a-C:H film. The I-D/I-G ratio about 0.71 suggests a hydrogenated amorphous carbon film (a-C:H). The deconvoluted XPS spectra of C 1s peaks disclose the presence of three main components: Cl associated to C=C bonds in one sp(2) hybridization as well as C2 associated to sp(3) hybridization. The impedance results showed that a-C:H film acts as a protective barrier against the corrosion process. Regardless of the cell population, when the total apoptosis of osteoblastic cells or PBMCs was evaluated, only cells cultured on the alloy without a-C:H had significant induction of the apoptotic process. Moreover, only PBMCs cultured on the alloy without a-C:H had significant induction of inflammatory and anti-inflammatory cytokines that characterize immune activation.